Surf1, associated with Leigh syndrome in humans, is a heme-binding protein in bacterial oxidase biogenesis.

Abstract

Biogenesis of mitochondrial cytochrome c oxidase (COX) relies on a large number of assembly factors, among them the transmembrane protein Surf1. The loss of human Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder caused by severe COX deficiency. In the bacterium Paracoccus denitrificans, two homologous proteins, Surf1c and Surf1q, were identified, which we characterize in the present study. When coexpressed in Escherichia coli together with enzymes for heme a synthesis, the bacterial Surf1 proteins bind heme a in vivo. Using redox difference spectroscopy and isothermal titration calorimetry, the binding of the heme cofactor to purified apo-Surf1c and apo-Surf1q is quantified: Each of the Paracoccus proteins binds heme a in a 1:1 stoichiometry and with Kd values in the submicromolar range. In addition, we identify a conserved histidine as a residue crucial for heme binding. Contrary to most earlier concepts, these data support a direct role of Surf1 in heme a cofactor insertion into COX subunit I by providing a protein-bound heme a pool.

Gel electrophoresis of purified Surf1 proteins.A, Coomassie-stained SDS-polyacrylamide gel of Surf1 proteins after immobilized metal ion affinity chromatography purification. Approximately 3 μg of protein was loaded per lane. B, heme- and Ponceau-stained blot of a lithium dodecyl sulfate (LDS)-polyacrylamide gel of purified Surf1 proteins. Five micrograms were loaded per lane, and the sample buffer contained either lithium dodecyl sulfate or SDS as indicated under the bars. Apo-Surf1c and apo-Surf1q were expressed in E. coli in the absence of the heme a-synthesizing enzymes, while Surf1c and Surf1q as well as the respective mutants were produced in their presence.

Spectra of oxidized heme a in the absence and presence of Surf1. Heme a spectra were recorded at a concentration of 3.75 μm in the Soret region in 20 mm phosphate, 0.02% n-dodecyl-β-d-maltoside, and 5% dimethyl sulfoxide. The following additions were made: A, buffer (solid line), apo-Surf1c (dashed line), and Surf1c mutant H193A (dotted line); B, buffer (solid line), apo-Surf1q (dashed line), and Surf1q mutant H202A (dotted line). Purified Surf1 proteins were added to a final concentration of 20 μm.

ITC titrations of Surf1 proteins and heme a.A, titration of heme a in the sample cell with wild-type Surf1q protein in the syringe. Top panel, raw heating power data. The first peak represents a small preinjection (5 μl) that is omitted in the integrated data. Bottom panel, data after peak integration and concentration normalization. B, isotherms for binding of heme a in the syringe to wild-type Surf1c (filled squares) or Surf1c H193A (open squares) in the sample cell. The curve is the fit of the data to a single-site binding model; for measurement conditions, see Table 1.

Hypothetical role of Surf1 in co-translational incorporation of heme a into COX SU I. Membrane-spanning Surf1 (yellow) in its unloaded apo form interacts with heme a synthase (HAS, green) to receive the heme group. Surf1 is suggested to interact directly with the nascent COX SU I polypeptide (orange) emerging from the ribosome (blue) eventually to receive both of its heme groups that are present in its fully folded form. Multiple copies of Surf1 depicted here denote its presumed heme a pool function.